Acute lung injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) are common, devastating clinical syndromes that affect large numbers of (200,000 cases in the US per year) and have approximately 30% mortality with the current standard of care. We have developed a highly effective treatment for this disease in mouse and pig models that uses the ubiquitous overexperssion of the ?1 subunit of the Na+,K+-ATPase to increase alveolar fluid clearance from the previously injured lung. Our experiments show that this treatment not only improves edema resolution (and lung function and survival), but also improves alveolar epithelial/ endothelial barrier function by upregulating tight junction complexes. Highly efficient and safe gene delivery is carried out using electroporation, the application of brief synchronized square wave electric pulses across the chest following naked DNA delivery by aspiration. The procedure causes no trauma, no inflammation, no lung injury, no cardiac dysfunction, and uses less than 0.1 J/kg of energy in 50 kg healthy or septic pigs. We have had no deaths from transthoracic electroporation at optimal field strengths in over 90 healthy and 60 septic pigs with ARDS. We have found that MRCK?, a serine/threonine-protein kinase and a downstream effector of Cdc42 for cytoskeletal reorganization, is activated by ?1 overexpression and is needed for the increased activity/abundance of tight junction proteins caused by ?1. We have shown that these two proteins interact, that the ?1 subunit activates MRCK?, that inhibition or genetic silencing of MRCK? in alveolar type I epithelial cells abrogates the ability of ?1 overexpression to increase tight junction abundance and activity in cultured cells, and that overexpression of MRCK? improves barrier properties in cultured alveolar type I epithelial cells. While ?1 overexpression increases edema clearance and barrier function, we do not know which activity plays the predominant role in its treatment ability. Further, the identification of MRCK? may provide a new target for treatment of ALI/ARDS. We have also found that the miRNA miR-181a that has been reported to be significantly increased in the serum of ARDS patients, targets the 3'UTRs of both the Na+,K+-ATPase ?1 subunit (but not any other Na+,K+-ATPase subunit) and MRCK?. Inhibition of this miRNA by transfection of an antagomer increases expression of both the ?1 subunit and MRCK? in cells. Our studies will also test whether modulation of miR-181a can increase both the Na+,K+-ATPase ?1 subunit and MRCK? to aid resolution of lung injury in mouse ALI/ARDS models. We will use novel cyclic amphipathic peptide nanoparticles for RNA delivery that we have used successfully in cells and the mouse lung. The aims are to (1) determine whether improved alveolar fluid clearance is the primary mechanism by which gene transfer of the Na+,K+-ATPase treats ALI/ARDS; (2) test whether induction of barrier function by gene transfer of MRCK? can mediate protection and/or treatment of ALI/ARDS in mice; and (3) determine whether gene transfer of an miR-181a inhibitor alone can treat ALI/ARDS or further enhance Na+,K+-ATPase gene transfer-mediated treatment in mice.